Limits...
PP2A/B56 and GSK3/Ras suppress PKB activity during Dictyostelium chemotaxis.

Rodriguez Pino M, Castillo B, Kim B, Kim LW - Mol. Biol. Cell (2015)

Bottom Line: Our follow-up research uncovered that B56 preferentially associated with GDP forms of RasC and RasD, but not with RasG in vitro, and psrA(-) cells displayed inefficient activation of multiple Ras species, decreased random motility, and inefficient chemotaxis toward cAMP and folic acid gradient.Surprisingly, psrA(-) cells displayed aberrantly high basal and poststimulus phosphorylation of Dictyostelium protein kinase B (PKB) kinase family member PKBR1 and PKB substrates.In summary, B56 constitutes inhibitory circuits for PKBA and PKBR1 and thus heavily affects Dictyostelium chemotaxis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Florida International University, Miami, FL 33199.

No MeSH data available.


Related in: MedlinePlus

Cells treated with LiCl exhibited increased PKBR1 activation. (A) Wt cells treated with GSK3 inhibitor, LiCl, showed increased poststimulus phosphorylation levels of PKBR1 compared with untreated cells: Wt cells treated with LiCl exhibited ∼30% increase in the basal level of PKBR1 activity and an approximately twofold increase in the level of PKBR1 phosphorylation after cAMP stimulation compared with nontreated cells (**, p < 0.01). The phospho-PKBR1 levels were normalized to Coomassie-stained total proteins. (B) LiCl-treated psrA− cells exhibited even higher levels of basal phosphorylation of PKBR1 compared with nontreated psrA− cells (p < 0.05). At 5-s poststimulus, the phosphorylation level of LiCl-treated psrA− cells was consistently higher than the control (**, p < 0.01). The phospho-PKBR1 levels were normalized to Coomassie-stained total proteins. (C) Unlike Wt or psrA− cells, cells lacking GSK3 showed no significant differences in PKBR1 phosphorylation in response to LiCl treatment (+, p > 0.05). The phospho-PKBR1 levels were normalized to Coomassie-stained total proteins. Error bars represent SD.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4666131&req=5

Figure 8: Cells treated with LiCl exhibited increased PKBR1 activation. (A) Wt cells treated with GSK3 inhibitor, LiCl, showed increased poststimulus phosphorylation levels of PKBR1 compared with untreated cells: Wt cells treated with LiCl exhibited ∼30% increase in the basal level of PKBR1 activity and an approximately twofold increase in the level of PKBR1 phosphorylation after cAMP stimulation compared with nontreated cells (**, p < 0.01). The phospho-PKBR1 levels were normalized to Coomassie-stained total proteins. (B) LiCl-treated psrA− cells exhibited even higher levels of basal phosphorylation of PKBR1 compared with nontreated psrA− cells (p < 0.05). At 5-s poststimulus, the phosphorylation level of LiCl-treated psrA− cells was consistently higher than the control (**, p < 0.01). The phospho-PKBR1 levels were normalized to Coomassie-stained total proteins. (C) Unlike Wt or psrA− cells, cells lacking GSK3 showed no significant differences in PKBR1 phosphorylation in response to LiCl treatment (+, p > 0.05). The phospho-PKBR1 levels were normalized to Coomassie-stained total proteins. Error bars represent SD.

Mentions: Consistent with the phenotype of dnGSK3-expressing cells, LiCl-treated wild-type cells displayed a statistically significant increase in the basal PKBR1 level (**p < 0.01) as well as cAMP-induced PKBR1 activation (Figure 8A). psrA− cells pretreated with LiCl also exhibited higher basal and cAMP-induced PKBR1 phosphorylation (Figure 8B). gsk3− cells, in contrast to the cells expressing dnGSK3 or treated with LiCl, showed no PKBR1 phosphorylation regardless of LiCl treatment (Figure 8C), indicating that the effect of LiCl is GSK3 dependent. Thus the high GSK3 activity in psrA− cells (Lee et al., 2008) is unlikely to be the main cause for the high PKBR1 activity in psrA− cells. We also noticed that, unlike dnGSK3-expressing psrA− cells, psrA− cells treated with LiCl demonstrated no increase in PKBA. Although LiCl can clearly inhibit GSK3, it can also block cAMP-dependent PIP3 generation and thus insulate PKBA activation (King et al., 2009). The molecular nature of LiCl-mediated inhibition of cAMP-induced PKBA activation is currently unclear, but in any case, PP2A/B56 is not necessary for the LiCl effect.


PP2A/B56 and GSK3/Ras suppress PKB activity during Dictyostelium chemotaxis.

Rodriguez Pino M, Castillo B, Kim B, Kim LW - Mol. Biol. Cell (2015)

Cells treated with LiCl exhibited increased PKBR1 activation. (A) Wt cells treated with GSK3 inhibitor, LiCl, showed increased poststimulus phosphorylation levels of PKBR1 compared with untreated cells: Wt cells treated with LiCl exhibited ∼30% increase in the basal level of PKBR1 activity and an approximately twofold increase in the level of PKBR1 phosphorylation after cAMP stimulation compared with nontreated cells (**, p < 0.01). The phospho-PKBR1 levels were normalized to Coomassie-stained total proteins. (B) LiCl-treated psrA− cells exhibited even higher levels of basal phosphorylation of PKBR1 compared with nontreated psrA− cells (p < 0.05). At 5-s poststimulus, the phosphorylation level of LiCl-treated psrA− cells was consistently higher than the control (**, p < 0.01). The phospho-PKBR1 levels were normalized to Coomassie-stained total proteins. (C) Unlike Wt or psrA− cells, cells lacking GSK3 showed no significant differences in PKBR1 phosphorylation in response to LiCl treatment (+, p > 0.05). The phospho-PKBR1 levels were normalized to Coomassie-stained total proteins. Error bars represent SD.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4666131&req=5

Figure 8: Cells treated with LiCl exhibited increased PKBR1 activation. (A) Wt cells treated with GSK3 inhibitor, LiCl, showed increased poststimulus phosphorylation levels of PKBR1 compared with untreated cells: Wt cells treated with LiCl exhibited ∼30% increase in the basal level of PKBR1 activity and an approximately twofold increase in the level of PKBR1 phosphorylation after cAMP stimulation compared with nontreated cells (**, p < 0.01). The phospho-PKBR1 levels were normalized to Coomassie-stained total proteins. (B) LiCl-treated psrA− cells exhibited even higher levels of basal phosphorylation of PKBR1 compared with nontreated psrA− cells (p < 0.05). At 5-s poststimulus, the phosphorylation level of LiCl-treated psrA− cells was consistently higher than the control (**, p < 0.01). The phospho-PKBR1 levels were normalized to Coomassie-stained total proteins. (C) Unlike Wt or psrA− cells, cells lacking GSK3 showed no significant differences in PKBR1 phosphorylation in response to LiCl treatment (+, p > 0.05). The phospho-PKBR1 levels were normalized to Coomassie-stained total proteins. Error bars represent SD.
Mentions: Consistent with the phenotype of dnGSK3-expressing cells, LiCl-treated wild-type cells displayed a statistically significant increase in the basal PKBR1 level (**p < 0.01) as well as cAMP-induced PKBR1 activation (Figure 8A). psrA− cells pretreated with LiCl also exhibited higher basal and cAMP-induced PKBR1 phosphorylation (Figure 8B). gsk3− cells, in contrast to the cells expressing dnGSK3 or treated with LiCl, showed no PKBR1 phosphorylation regardless of LiCl treatment (Figure 8C), indicating that the effect of LiCl is GSK3 dependent. Thus the high GSK3 activity in psrA− cells (Lee et al., 2008) is unlikely to be the main cause for the high PKBR1 activity in psrA− cells. We also noticed that, unlike dnGSK3-expressing psrA− cells, psrA− cells treated with LiCl demonstrated no increase in PKBA. Although LiCl can clearly inhibit GSK3, it can also block cAMP-dependent PIP3 generation and thus insulate PKBA activation (King et al., 2009). The molecular nature of LiCl-mediated inhibition of cAMP-induced PKBA activation is currently unclear, but in any case, PP2A/B56 is not necessary for the LiCl effect.

Bottom Line: Our follow-up research uncovered that B56 preferentially associated with GDP forms of RasC and RasD, but not with RasG in vitro, and psrA(-) cells displayed inefficient activation of multiple Ras species, decreased random motility, and inefficient chemotaxis toward cAMP and folic acid gradient.Surprisingly, psrA(-) cells displayed aberrantly high basal and poststimulus phosphorylation of Dictyostelium protein kinase B (PKB) kinase family member PKBR1 and PKB substrates.In summary, B56 constitutes inhibitory circuits for PKBA and PKBR1 and thus heavily affects Dictyostelium chemotaxis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Florida International University, Miami, FL 33199.

No MeSH data available.


Related in: MedlinePlus